Impurities

Evaluating Impurities in Drugs

Part III of III

ADAM GAuLT/ OJO IMAGeS/GeTTy IMAGeS

Kashyap R. Wadekar, Ponnaiah Ravi, Mitali Bhalme, S. Srinivasa Rao,

K. Vigneshwar Reddy, L Sampath Kumar, and E. Balasubrahmanyam

A process generating an inordinate amount of

impurities may be optimized to decrease the levels
of impurities before scale-up. Once the structure of
the impurity or impurities is known, the mechanism
of formation generally can be elucidated, and
conditions to avoid these impurities may be
designed. In Part III of this article, the authors
examine various degradation routes of APIs,
impurities arising from APIexcipient interaction
during formulation, metabolite impurities,
various analytical methodologies to measure
impurity levels, and ways to control impurities in
pharmaceuticals.

Kashyap R. Wadekar, PhD,* is a research

scientist (II), Ponnaiah Ravi, PhD, is senior vicepresident of R&D, Mitali Bhalme, PhD, is an associate
research scientist, S. Srinivasa Rao is a research
associate, K. Vigneshwar Reddy is a research
associate, L. Sampath Kumar is a research chemist,
and E. Balasubrahmanyam is a research chemist, all
with Neuland Laboratories, 204 Meridian Plaza, 6-3854/1, Ameerpet, Hyderabad, India, tel. 91 40 30211600,
kashyapwadekar@neulandlabs.com.
*To whom all correspondence should be addressed.
Submitted: Sept. 19, 2011; Accepted Nov. 28, 2011.

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ontrolling and monitoring impurities in APIs and finished

drug products is a crucial issue in drug development and
manufacturing. Part I of this article, published in the February 2012 issue of Pharmaceutical Technology, discussed
the various types of and sources of impurities with specific case
studies (1). Part II, published in the March 2012 issue, examined
chiral, polymorphic, and genotoxic impurities (2). In Part III, the
authors examine various degradation routes of APIs, impurities
arising from APIexcipient interaction during formulation, metabolite impurities, various analytical methodologies to measure
impurity levels, and ways to control impurities in pharmaceuticals.

Definition of impurity
The term impurity reflects unwanted chemicals that are present
in APIs or that develop during formulation or upon aging of
the API in the formulated drug product. The presence of such
unwanted material, even in small amounts, could affect the efficacy and safety of pharmaceutical products. Several guidelines
from the International Conference on Harmonization (ICH) address impurities in new drug substances, drug products, and residual solvents (36). As per the ICH guidelines on impurities in
new drug products, impurities present below a 0.1% level do not
need to be qualified unless the potential impurities are expected
to be unusually potent or toxic (5). In all other cases, impurities
should be qualified. If the impurities exceed the threshold limits
and data are not available to qualify the proposed specification
level, studies to obtain such data may be required. Several recent
articles describe a designed approach and guidelines for isolation and identification of process-related impurities and degradation products using mass spectrometry, nuclear magnetic
resonance (NMR) spectroscopy, high-performance liquid chromatography (HPLC), and Fourier transform infrared (FTIR)
spectroscopy for pharmaceutical substances (79).

Degradation-related impurities
Degradation products are compounds produced by decomposition of the material of interest or active ingredient. Several
impurities may result because of API degradation or other interaction on storage, so stability studies need to be conducted
to ensure drug product safety (10). Hydrochlorothiazide (see
Figure 1) is a classical example of a degradation impurity. It
has a known degradation pathway through which it degrades

Impurities
Figure 1: Degradation of hydrochlorothiazide.

NH2

O2S

O
S

NH2

O
S

O2 S

-(CH2O)n

N
H

CI

NH2

CI

Hydrochlorothiazide

Disulfonamide degradation product

Figure 2: Reaction scheme for mirtazapine impurity. Ph. Eur is the European Pharmacopoeia. DMF
is dimethylformamide. EtOAc is ethyl acetate.

O
O

H
N

+
N

DMF, EtOAc

O
N

KF

CI

O
N

1
NaBH4
Ethanol, water
acetone, EtOAc
OH
N

H2SO4
N

N
O
N

ALL fIGuReS ARe cOuRTeSy Of THe AuTHORS

Mirtazapine Impurity C (Ph. Eur)

to the starting material as disulfonamide in its synthesis.

Degradation products could result from the synthesis itself,
storage, formulation of the dosage form, and aging (11). These
degradation pathways are further discussed.
Synthesis-related impurities. Impurities in a drug substance or a
new chemical entity originate mainly during the synthetic process
from raw materials, solvents, intermediates, and byproducts. The
raw materials are generally manufactured to much lower purity
requirements than a drug substance, and thus, it is easy to understand why they can contain a number of components that can in
turn affect the purity of the drug substance.
1-Methyl-3-phenyl piperazine (see Figure 2) is present as an unreacted starting material that competes in all the stages eventually
leading to the impurity keto-piperazine derivative of mirtazapine
(see Impurity C, Figure 2).
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N
O
N

Formulation-related
impurities. Several impurities
in a drug product or API can
arise from interactions with
excipients used to formulate
the drug product. In the process of formulation, a drug
substance is subjected to various conditions that can lead
to its degradation or other
deleterious reactions. For example, if heat is used for drying or for other reasons, it can
facilitate degradation of thermally labile drug substances.
Solutions and suspensions are
potentially prone to degradation due to hydrolysis or solvolysis. These reactions also
can occur in the dosage form
at solid state, such as in the
case of capsules and tablets,
when water or another solvent
has been used for granulation.
There are two typical conditions in solid- and solutionstate degradation studies.
Typical conditions for the API
in a solid state might be 80 C,
75% relative humidity (RH);
60 C at ambient RH; 40 C
at 75% RH; and light irradiation. Typical conditions for
an API in the solution state
might be: pH 19 in buffered
media; with peroxide and/
or free-radical initiator; and
light irradiation.
Figure 3 shows the degradation pathway of ketorolac in the

solid and solution states (1214).

Dosage form-related impurities. Impurities related to the dosage
form are significant because many times precipitation of the
main ingredient requires various factors, such as pH or leaching,
to be altered (15). For example, the precipitation of imipramine
hydrochloride with sodium bisulfite requires a subsequent pH
alteration of lidocaine hydrochloride solution in the presence
of 5% dextrose in saline.
Method-related impurities. A known impurity,1-(2,6dichlorophenyl)indolin-2-one is formed in the diclofenac sodium ampuls. Formation of this impurity depends on the initial
pH of the preparation and the conditions of sterilization (i.e.,
autoclave method, 123 C 2 C) that enforces the intermolecular cyclic reaction of diclofenac sodium, forming indolineone
derivative and sodium hydroxide (16).

Impurities
Figure 3: Degradation pathway of ketorolac.

- CO2

O
O

decarboxy analog

O2
OH

N
O

ketorolac

NH2-C-(CH2OH)3

OH

1-hydroxy analog

O2

solid state only

O
N
O
amide

NHC(CH2OH)3

O
1-keto analog

Environmental-related impurities. EnvironTable I: Effect of interactions among ingredients

mental-related impurities may result from
the following:
Active ingredient
Pharmaceutical aid
Effect
Temperature. Many heat-labile comKanamycin
Honey; sugar syrup
Loss of activity at room temperature
pounds, when subjected to extreme tem2% polyoxy ethylene ester
Change in pH, degradation of active
Cholecalciferol
perature, lose their stability. Keeping this
surfactant; polysorbate
ingredient.
in mind, extreme care should be exerCalcium or magnesium or
Tetracyclines
Complexation
cised to prevent them from degradation.
metal ions
Light (ultraviolet light). Exposure to light reBromine; Chloride-based
Formed different soluble halides of
sults in a photolytic reaction. Several studThiomersal
compounds; Iodide-based
cationic mercury compounds
ies reported that ergometrine and ergometcompounds
rine injections are unstable under tropical
Adrenaline
Boric acid;povidone
Stabilization
condition such as light and heat (1719).
Humidity. Humidity is one of the important
factors when working with hygroscopic compounds. Humidity Photolysis. Photolytic cleavage on aging products occurs with
can be deleterious to bulk powders and formulated solid dosAPIs or drug products that are prone to degradation on exage forms. Well-know examples are ranitidine and aspirin (19).
posure to UV light. For example, the ophthalmic formulation
Impurities on aging. Generally, a longer stay on the shelf increases
of ciprofloxacin drops 0.3%, when exposed to UV light and
the possibility that impurities will occur. Such impurities can be
undergoes photolysis to form ethylene diamine, an analog of
caused by several interactions as further described.
ciprofloxacin (22).
Interaction among ingredients. Vitamins are highly prone to Decarboxylation. Carboxylic acid (COOH) tends to lose
instability after aging. For example, the presence of nicotincarbon dioxide from carboxyl groups when heated. For
amide containing four vitamins (nicotinamide, pyridoxine,
instance, a photoreaction of a rufloxacin enteric tablet
riboflavin, and thiamin) caused the degradation of thiamin to
coated with cellulose acetate phthalate and subcoated with
a substandard level during a one-year shelf life (20). Table I lists
calcium carbonate causes hydrolysis of cellulose acetate
some examples of interactions among ingredients.
phthalate. This reaction liberates acetic acid, which on re Hydrolysis. Many drugs are derivatives of carboxylic acids or
acting with calcium carbonate, produces carbon dioxide
contain functional groups susceptible to acidbase hydrolysis
as a byproduct.
(e.g., aspirin, atropine, and chloramphenical).
pH. It is well understood that pH, particularly extreme levels
Oxidation. In pharmaceuticals, the most common form of oxiof pH, can encourage hydrolysis of the API when ionized in
dative decomposition is auto-oxidation through a free-radical
an aqueous solution. This situation necessitates buffer control
chain process. Drugs that are prone to oxidation include methif such a dosage form is required.
otrexate, adinazolam, catecholamine, conjugated dienes (i.e., Packaging materials. Impurities may result from packaging
vitamin A), and nitroso and nitrite derivatives. Olanzapine is
materials (i.e., containers and closures (23).
especially prone to oxidative degradation in the presence of
Two impurities in olanzapine have been identified as
oxygen (see Figure 4) (21).
1 and 2 (see Figure 4) (24). The structures indicate that the two
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impurities are degradation products resulting from oxidation of the thiophene

ring of olanzapine.
From a regulatory perspective, forced
degradation provides data to support the
following (25):
Identification of possible degradants
Degradation pathways and intrinsic
stability of the drug molecule
Validation of stability for indicating
analytical procedures
Facilitation of the development of analytical methods to evaluate stability
Understanding the degradation of
the API to a rational product.
Screening for possible formation of
potential genotoxins.
Various issues are addressed in regulatory guidance (36). Some key issues are:
Forced degradation is typically carried out using one batch of material.
Forced-degradation conditions are
more severe than accelerated stability
testing, such as 50 C; 75% RH; light
conditions exceeding ICH standards;
high and low pH; and oxidation.
Photostability should be an integral
part of forced-degradation study
design (10).
Degradation products that do not
form in accelerated or long-term
stability may not have to be isolated
or have their structure determined.
Mass balance should be considered.
Various issues are not addressed in
regulatory guidance (3-6). Some key
issues not addressed are:
Exact experimental conditions
(temperatures, duration, and extent
of degradation)
Experimental design (left to the applicants discretion).

Metabolite impurities
Metabolite impurities are byproducts
formed in the body after a drug substance is ingested. During metabolism,
the API and drug product in the body
are exposed to various enzymes, from
which metabolite impurities can be
formed (2634). Drug metabolism is
traditionally divided into two phases:
metabolic (i.e., hepatic) clearance and
the Phase I and Phase II process. The
division is based on the observation that
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a drug substance first undergoes oxidative attack (e.g., benzene

to phenol), and the newly introduced hydroxyl function will
undergo glucouronidation (e.g., phenol to phenyl glucouronic
acid). Some metabolites are formed as impurities during the development of a process. Control of these process-related metabolite impurities in the final API may not be necessary if control
of other metabolites has already occurred and taken into consideration. Tightening the limits, therefore, may not be needed.
Examples are asenapine N-oxide, asenapine desmethyl,
and ciprofoxacin ethyl diamino impurity, which are formed
as process impurities, but are also metabolites of the same
process (see Figure 5). It put forth a question whether limiting
such a metabolite impurity in the final API is still required.

Select analytical methodologies

The development of a new drug mandates that meaningful
and reliable analytical data be generated at various steps of
drug development. The drug also should exhibit excellent
stability throughout its shelf-life. To meet these requirements, methodologies need to be developed that are sensitive
enough to measure low levels of impurities. This need has
led to analytical methods that are suitable for determining
trace and ultra-trace levels (i.e., submicrogram) quantities
of various chemical entities (3539). Various methods are
available for monitoring impurities.

Spectroscopic methods. Various spectroscopic methods can

be used for characterization of impurities, such as UV-visible spectroscopy, FTIR spectroscopy, NMR spectroscopy,
and mass spectrometry (MS).
Separation methods. Various separation methods can be
used, including thin-layer chromatography (TLC), gas
chromatography (GC), HPLC, capillary electrophoresis
(CE), and supercritical fluid chromatography (SFC). A review of these methods is provided in the literature (39).
CE is an electrophoretic method that is frequently lumped
with chromatographic methods because it shares many of
the common requirements of chromatography. A broad
range of compounds can be resolved using TLC by using
different plates and mobile phases. GC is a useful technique
for quantification. It can provide the desired resolution,
selectivity, and ease of quantification. This technique is
useful for organic volatile impurities. SFC offers some of
the advantages of GC in terms of detection and HPLC in
terms of separation.
Hyphenated methods. The following hyphenated methods can
be used effectively to monitor impurities: GCMS; liquid chromatography (LC)MS; LCdiode-array detection (DAD)MS;
LCNMR; LCDADNMRMS; and LCMSMS.
Isolating impurities. It is often necessary to isolate impurities because the instrumental methods are not available or

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83

Impurities
Figure 4: Olanzapine impurities due to air, heating, and formulation.

N
N

air
heating

N
H

N
H

Olanzapine

N
H

N
N

Olanzapine lactam Impurity.

Olanzapine thiolactam Impurity.

Ideally, an impurity profile should show all impurities in a single

format to allow monitoring of any variation in the profile. The
driving forces for studying an impurity profile are quality considerations and regulatory requirements.
Samples to be profiled. Impurity profiling should be done
for APIs, process check of the synthesis or formulation, and
final drug product.
Components in an impurity profile. Ideally, an impurity profile
should show synthesis-related impurities, formulation-related
impurities, degradation products, and interaction products.
Crucial factors for controlling impurities in APIs. Several factors are
important in controlling impurities in APIs as further outlined.
Crystallization. The size of crystals not only determines the
quality, but also the stability of the drug. During crystallization, fine crystals should be formed to prevent entrapment of
minute amounts of chemicals from the mother liquor, which
in turn causes degradation of the drug.
Wet-cake washing. Many unwanted chemicals, including residual
solvents, could be removed by thorough washing of the wet cake,
which if not done correctly, could lead to retention of solvents and
impurities in the cake.
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Hydroxymethylidine thione

Impurity profiling

OH

NH

further confirmation is needed. The following methods have

been used for isolation of impurities: solid-phase extraction,
liquidliquid extraction, accelerated solvent extraction, supercritical fluid extraction, column chromatography, flash
chromatography, TLC, HPLC, CE, and SFC.

O
NH

84

Acetoxymethylidine thione

Drying. Use of vacuum dryer or a fluid-bed dryer is always advisable in comparison to a tray dryer. Use of the former reduces
drying time and brings about uniform drying, which is helpful
in drying sensitive drug substances.
Appropriate packaging. The packing of bulk drugs should be
based upon their nature and sensitivity. Light-sensitive products should be packed in light protective packing. Use of opaque
containers for ciprofloxacin eye-drops preparations protects the
active ingredients from photodegradation (22). Use of ampuls
with either black carbon paper or aluminum foil for ergometrine
produced negligible degradation (40). It is important to determine the most appropriate container-closure system.
Production methods based on stability studies. A manufacturer of a
bulk drug should perform a detailed investigation of the process,
including stability studies while finalizing the method of preparation. For example, for producing diclofenac sodium injections,
the aseptic filtration process is better than the autoclave method
that produces the impurity (16).
Measures by pharmacopoeias. Pharmacopoeias should take
steps to incorporate impurity limits for drug substances
made from a raw material in which that particular impurity is controlled. It becomes convenient for the users if the
impurity limit is mentioned in the dosage forms.

Conclusion
Parts I, II, and III of this article discussed the types, origin,
causes, chemistry, and impact of impurities in APIs and drug
products (1, 2). Parts I and II explained how, when, and why

Impurities
Figure 5: Process impurities, thermal decomposition impurities, and metabolites of asenapine. CAS No. is Chemical Abstracts Service number.
O
CI
H

H
CI

N
H

Desmethyl asenapine
CAS No 128915-56-0
Metabolite
Process impurity

H
N

Deschloro asenapine
Process impurity

Cis-asenapine
Process impurity

CI
H
O

O
O

CI
H
O

COOH
O

Trans-lactam
Process impurity

Cis-lactam
Process impurity

COOH

CI

CI
Asenapine maleate

H
N

O
CI

CI

CI

*CAS No 129385-60-0

*CAS No 129385-61-1

Asenapine N-oxide
CAS No 128949-51-9
Metabolite

*CAS No 129385-59-7

* Thermal decomposition impurities

impurities are formed. This article, Part III, highlighted the

degradation-related, formulation-related, and metabolite impurities, the various analytical techniques available for their
identification and separation, and crucial factors that are to
be controlled while preparing bulk drugs.

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